Modified interleukin-1beta converting enzyme with increased stability

ABSTRACT

Modified forms of human interleukin-1β converting enzyme (ICE) that display proteolytic activity and, furthermore, have increased stability compared to unmodified human ICE are disclosed. Nucleic acid molecules encoding a modified p10 subunit of ICE, and recombinant vectors and host cells incorporating such nucleic acid molecules, are also disclosed. A modified ICE protein of the invention can be used to cleave proteolytically ICE substrates and to identify modulators of ICE activity in screening assays. Moreover, due to its enhanced stability, the modified ICE of the invention is particularly suitable for use in the preparation of ICE crystals for X-ray crystallography.

BACKGROUND OF THE INVENTION

[0001] Interleukin-1 is a cytokine having a broad spectrum of biologicalactivities (for reviews, see e.g., Dinarello, C. A. and Wolff, S. M.(1993) New Engl. J. Med. 328:106-113; and Dinarello, C. A. (1993) Trendsin Pharmacol. Sci. 14:155-159). IL-1 consists of two structurallyrelated polypeptides, interleukin-1α (IL-1α) and interleukin-1β (IL-1β).The two forms of IL-1 are encoded by different genes and have only27-33% amino acid identity but they interact with the same receptor andhave similar biological activities. Included among the biologicalfunctions attributed to IL-1 are induction of fever, sleep, anorexia andhypotension. IL-1is also involved in the pathophysiology of inflammatoryand autoimmune diseases, including rheumatoid arthritis, septic shock,inflammatory bowel disease and insulin dependent diabetes mellitus.IL-1α has been specifically implicated in the pathophysiology ofpsoriasis. IL-1 is also thought to play a role in immune responses toinfectious agents and in the pathogenesis of myeloid leukemias.

[0002] IL-1α and IL-1β are both synthesized as approximately 31 kDaprecursor molecules that are subsequently processed to a mature form ofapproximately 17 kDa. IL-1α and IL-1β differ in that the precursor formof IL-1α (preIL-1α) is biologically active and most of the matureIL-1α(matIL-1α) remains cell-associated, whereas the precursor form ofIL-1β (preIL-1β) must be cleaved to its mature form to become active andthe mature form of IL-1β (matIL-1β) is secreted from the cell. Onlycertain cell types process preIL-1β and secrete matIL-1β. Monocytes andmacrophages are the most efficient producers and secretors of IL-1β,which is the most abundant form of IL-1 produced upon activation ofthese cell types.

[0003] Interleukin-1β converting enzyme (ICE) is a cytoplasmic cysteineprotease required for generating the bioactive form of theinterleukin-1β cytokine from its inactive precursor (Black, R. A. et al.(1988) J. Biol. Chem. 263:9437-9442; Kostura, M. J. et al. (1989) Proc.Natl. Acad. Sci. USA 86:5227-5231; Thornberry et al. (1992) Nature356:768-774; Ceretti, D. P. et al. (1992) Science 256:97-100). ICE is amember of a family of cysteine proteases with shared homology. Othermembers of this family have been implicated in apoptosis, such as ced-3(Yuan, J. et al. (1993) Cell 75:641-652), Nedd2 (Kumar, S. et al. (1992)Biochem. Biophys. Res. Commun. 185:1155-1161; Kumar, S. et al. (1994)Genes Dev. 8:1613-1626), CPP32 (Fernandes-Alnemri, T. et al. (1994) J.Biol. Chem. 269:30761-30764), Ich-1 (Wang, L. et al. (1994) Cell78:739-750) and Ich-2 (Kamens, J. et al. (1995) J. Biol Chem.270:15250-15256; Faucheu, C. et al. (1995) EMBO J. 14:1914-1922). Thenecessity for ICE in the generation of bioactive IL-1β was demonstratedin mice in which the ICE gene had been functionally disrupted (Li, P. etal. (1995) Cell 80:401-411; Kuida. K. et al (1995) Science267:2000-2003). Although these animals are overtly normal, they have amajor defect in the production of mature IL-1-β after stimulation withlipopolysaccharide.

[0004] In vitro studies have demonstrated that ICE cleavesprointerleukin-1β at Asp₁₁₆-Ala₁₁₇ to release the fully active 17 kDaform (Black, supra; Kostura, supra). ICE also cleaves prointerleukin-1βat Asp₂₇-Ala₂₈ to release a 28 kDa form. Cleavage at these sites isdependent upon the presence of aspartic acid in the P1 position(Kostura, supra, Howard, A. et al. (1991) J. Immunol. 147:2964-2969;Griffin, P. R. et al. (1991) Int. J. Mass. Spectrom. Ion. Phys.111:131-149). However, an aspartic acid in the P1 position is notsufficient for ICE specificity. For example, several other proteinscontaining Asp-X bonds, including prointerleukin-1α, are not cleaved byICE (Howard, supra).

[0005] ICE itself undergoes maturational processing, possibly performedin vivo by ICE itself (Thornberry, N. A. et al. (1992) Nature356:768-774). Mature ICE is generated from a 404 amino acid precursorprotein by proteolytic removal of two fragments, the N-terminal 119amino acid “pro-domain” and the internal residues 298-316 (Thornberry,supra). Active ICE is therefore composed of two subunits, a 20 kDasubunit (p20) encompassing residues 120 to 297 and a 10 kDa subunit(p10) encompassing residues 317 to 404. The crystal structure of ICEindicates that ICE forms a tetrameric structure consisting of two p20and two p10 subunits (Walker, N. P. C. et al. (1994), Cell 78:343-352;Wilson, K. P. et al. (1994) Nature 370:270-275). The catalytic aminoacid residues of ICE are Cys-285 and His-237. The side chains of fouramino acid residues (Arg-179, Gln-283, Arg-341 and Ser-347) form the P1carboxylate binding pocket (Walker, supra; Wilson, supra).

[0006] Because of the apparently harmful role of IL-1 in many diseaseconditions, therapeutic strategies aimed at reducing the production oraction of IL-1 have been proposed. For example, one approach by which toinhibit matIL-1β production and secretion would be to block the activityof ICE with a specific ICE inhibitor. The ability to produce active ICEin vitro is therefore highly desirable to allow for the study of itsstructure and function. However, in vitro production of ICE can behampered by the instability of the protein, in particular as a result ofautocatalytic degradation which leads to inactive protein.

SUMMARY OF THE INVENTION

[0007] This invention provides modified human ICE proteins that retainthe proteolytic activity of unmodified human ICE and that exhibitincreased stability in vitro compared to modified ICE. One aspect of theinvention pertains to a modified form of ICE comprising an amino acidsequence wherein an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutantamino acid structure. This mutant amino acid structure is one that iscapable of forming a salt bridge with an amino acid corresponding toarginine at position 383 of unmodified ICE, such that the modified ICEexhibits proteolytic activity and has increased stability compared tounmodified ICE. The mutant amino acid structure that replaces the aminoacid corresponding, to Asp381 can be a natural amino acid or anon-natural amino acid. In the most preferred embodiment, the mutantamino acid structure is a glutamic acid residue. In other embodiments,the mutant amino acid structure is selected from the group consisting ofserine, threonine, asparagine and glutamine.

[0008] Another aspect of the invention pertains to nucleic acidmolecules encoding a modified p10 subunit of ICE. The modified p10subunit encoded by the nucleic acid molecule comprises an amino acidsequence wherein an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutantamino acid structure. The mutant amino acid structure is one that iscapable of forming a salt bridge with an amino acid corresponding toarginine at position 383 of unmodified ICE, such that the modified p10subunit associates with a p20 subunit to form a modified ICE thatretains proteolytic activity and exhibits increased stability comparedto unmodified ICE. In the most preferred embodiment, the p10 subunitencoded by the nucleic acid molecule has a glutamic acid residue at theposition corresponding to position 381 of unmodified ICE. In otherembodiments, the p10 subunit has a serine, threonine, asparagine orglutamine residue at the position corresponding to position 381 ofunmodified ICE.

[0009] A nucleic acid molecule of the invention encoding a modified p10subunit of ICE can be incorporated into a recombinant expression vector.In one embodiment, the recombinant expression vector encodes themodified p10 subunit of ICE (i e., about amino acids 317 to 404). Inanother embodiment, the recombinant expression vector encodes themodified p10 subunit of ICE and also encodes the p20 subunit of ICE(i.e., about amino acids 120-197). For example, in one embodiment, therecombinant expression vector encodes the p30 form of ICE, comprisingabout amino acids 120-404. This p30 form of ICE undergoes maturationalprocessing to produce a modified p10 subunit and a p20 subunit.

[0010] The recombinant expression vectors of the invention can beintroduced into host cells to produce modified ICE proteins. In oneembodiment, a modified p10 subunit is expressed recombinantly in a hostcell, denatured and refolded with a p20 subunit of ICE to form amodified ICE protein of the invention. In another embodiment, a modifiedp10 subunit is coexpressed with a p20 subunit in the same host cell(using either two separate expression vectors or one expression vectorencoding both the p10 and p20 subunits, such as a vector encoding p30),thereby producing a modified ICE protein of the invention.

[0011] The modified ICE proteins of the invention are cysteine proteasesthat exhibit proteolytic activity against ICE substrates. Accordingly, amodified ICE protein of the invention can be used to cleave an ICEsubstrate by contacting the ICE substrate with the modified ICE suchthat the ICE substrate is cleaved. Moreover, the modified ICE proteinsof the invention can be used in screening assays to identify modulators(e.g., inhibitors or stimulators) of ICE protease activity. Stillfurther, the enhanced stability of the modified ICE proteins of theinvention makes them particularly well-suited for the preparation ofcrystalline ICE for use in X-ray cystallographic analysis (e.g., forstructure-based design of ICE inhibitors).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a photograph of an SDS-polyacrylamide gel comparing E.coli-expressed wild-type N-His ICE protein and E. coli-expressedD381E-modified N-His ICE protein.

DETAILED DESCRIPTION OF THE INVENTION

[0013] This invention provides modified forms of human interleukin-1βconverting enzyme (ICE) which retain the proteolytic activity ofunmodified ICE yet also exhibit increased stability compared tounmodified human ICE. The invention is based, at least in part, on thediscovery that mutation of ICE at an aspartic acid at position 381(Asp-381) to glutamic acid (Glu-381) greatly increases the stability ofthe modified form of ICE (due to greatly reduced autocatalyticdegradation of the protein), while maintaining the proteolytic activityof the protein. In contrast, it was further discovered that mutation ofICE at Asp-381 to alanine (Ala-381) results in a modified ICE proteinthat, although stable, exhibits greatly reduced proteolytic activity.These results were then applied to an analysis of the crystal structureof human ICE. The crystal structure of ICE demonstrated that Asp-381forms a salt bridge with an arginine at position 383 (Arg-383). SinceGlu-381 maintains this salt bridge and Ala-381 does not, preferredmodifications in the modified ICE proteins of the invention aremodifications at a position corresponding to Asp-381 that retain thecapacity to form a salt bridge with an amino acid corresponding toArg-383.

[0014] Various aspects of the invention are described in further detailin the following subsections.

[0015] I. Modified ICE Proteins

[0016] One aspect of the invention pertains to modified ICE proteins. Asused herein, the term “unmodified ICE” refers to ICE proteins having theamino acid sequence of naturally-occurring human ICE. The term“unmodified ICE” includes the precursor form of ICE having the aminoacid sequence shown in SEQ ID NO: 2 (the correspondingnaturally-occurring nucleotide sequence that encodes unmodified humanICE is shown in SEQ ID NO: 1), as well as the processed mature form ofICE composed of p20 subunits (amino acids residues 120 to 297) and p10subunits (amino acid residues 317 to 404). As used herein, the terms“aspartic acid at position 381 of unmodified ICE” (or simply Asp-381)and “arginine at position 383 of unmodified ICE” (or simply Arg-383)refer to amino acid residues within the p10 subunit using the numberingbased on the full-length precursor form of ICE as shown in SEQ ID NO: 2.

[0017] As used herein, the term “modified ICE” refers to forms of ICEthat differ structurally from unmodified ICE. In particular, themodified ICE proteins of the invention comprise an amino acid sequencewherein an amino acid corresponding to aspartic acid at position 381 ofunmodified ICE (as shown in SEQ ID NO: 2) is replaced with a mutantamino acid structure, the mutant amino acid structure being capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE. The mutant amino acid structure isselected such that the modified ICE retains proteolytic activity andexhibits increased stability compared to unmodified ICE. The term“mutant amino acid structure” is intended to include natural amino acidsand non-natural amino acids. Non-natural amino acids include amino acidderivatives, analogues and mimetics. As used herein, a “derivative” ofan amino acid refers to a form of the amino acid in which one or morereactive groups on the compound have been derivatized with a substituentgroup. As used herein an “analogue” of an amino acid refers to acompound that retains chemical structures of the amino acid necessaryfor functional activity of the amino acid (e.g., formation of a saltbridge with Arg-383 in ICE) yet also contains certain chemicalstructures that differ from the amino acid. As used herein, a “mimetic”of an amino acid refers to a compound in that mimics the chemicalconformation of the amino acid.

[0018] Within the modified ICE proteins of the invention, an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE isreplaced with a mutant amino acid structure that is capable of forming asalt bridge with an amino acid corresponding to arginine at position 383of unmodified ICE. Mutant amino acid structures that are capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE can be determined by computer modelingusing the crystal structure of unmodified ICE (determination of thecrystal structure of ICE is described in Walker, N. P. C. et al. (1994),Cell 78:343-352; and Wilson, K. P. et al. (1994) Nature 370:270-275).According to the crystal structure of unmodified ICE, Asp-381 forms asalt bridge with Arg-383. It has now been discovered that substitutionof Asp-381 with glutamic acid, which retains the ability to form a saltbridge with Arg-383, results in a modified ICE that is bothproteolytically active and more stable than unmodified ICE. In contrast,substitution of Asp-381 with an alanine, which cannot form a salt bridgewith Arg-383, results in a modified ICE that, although stable has lostproteolytic activity, thereby demonstrating the necessity formaintenance of this salt bridge for effective proteolytic activity.

[0019] In a modified ICE of the invention, Asp-381 of unmodified ICE ispreferably substituted with another natural amino acid capable offorming a salt bridge with an amino acid corresponding to Arg-383. Inthe most preferred embodiment, Asp-381 is substituted with glutamicacid. In other embodiments, Asp-381 is substituted with an amino acidselected from the group consisting of serine, threonine, asparagine andglutamine. Other amino acids that can form a salt bridge with Arg-383include derivatives of aspartic acid and glutamic acid, such asβ-methylaspartic acid, γ-methylglutamic acid and β-methylglutamic acid,which may occur rarely in nature or which may be non-naturalderivatives. Additional non-natural amino acids that can be used includeanalogues or mimetics of aspartic acid and glutamic acid thatincorporate modifications into the peptide backbone of the ICE protein,such as an N-methyl aspartic acid or N-methyl glutamic acid. Moreover, amodified ICE protein of the invention can be modified at the amidelinkage between Asp-381 and Gly-382, for example, the amide linkage canbe substituted with an alkyl chain (thereby inhibiting proteolyticdigestion at this linkage).

[0020] The modified ICE proteins of the invention exhibit increasedstability compared to unmodified ICE. Preferably, the modified ICEprotein is at least 10% more stable than unmodified ICE. Morepreferably, the modified ICE protein is at least 25% more stable thanunmodified ICE. Even more preferably, the modified ICE is at least 50%more stable than unmodified ICE. Still more preferably, the modified ICEprotein is at least 75% more stable than unmodified ICE. The stabilityof modified and unmodified ICE proteins can be examined and quantifiedby direct or indirect means. For example, the degree of degradation ofmodified or unmodified ICE can be assessed directly and used as ameasure of the stability of the protein. The degree of degradation of anICE preparation can be visualized directly by examining the protein bystandard sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Samples containing a known amount of ICE protein (modifiedvs. unmodified) are electrophoresed on a standard SDS-PAGE gel and theprotein bands are visualized by standard methods (e.g., Coomassie bluestaining, silver staining and the like). Degradation of the ICEpreparation is evidenced by the appearance of lower molecular weightbreakdown products of the p10 subunit (i.e., protein band less than 10kDa in weight, such as a predominant 7 kDa band), as described furtherin Example 2, part C and illustrated in FIG. 1. This degradation can bequantified by quantitating the relative amounts of protein in the intactp10 band as compared to the lower molecular weight bands in the ICEpreparation. The relative amount of intact p10 in a modified ICEpreparation is then compared to the relative amount of intact p10 in anunmodified ICE preparation as a measure of the stability of the modifiedICE. For example, a modified ICE preparation in which the p10 band is50% less degraded than in the unmodified ICE preparation represents amodified ICE protein that is 50% more stable than an unmodified ICEprotein.

[0021] Alternatively, stability of the modified ICE proteins of theinvention can be assessed indirectly, for example by measuring theproteolytic activity of modified vs. unmodified ICE preparations overtime. A more stable ICE preparation exhibits greater proteolyticactivity over time than a less stable ICE preparation (see Example 2,parts A and B). Proteolytic activity can be assessed in a standard invitro proteolysis assay (such as described in Thornberry et al. (1992)Nature 356:768-774 and Example 2) using an appropriate ICE substrate.Examples of appropriate ICE substrates include the chromogenicpara-nitroanilide (pNA)-labeled peptide substrates,Acetyl-Tyr-Val-Ala-Asp-pNA (Ac-YVAD-pNA) and Acetyl-Asp-Glu-Val-Asp-pNA(Ac-DEVD-pNA), and the fluorogenic amino-4-methylcoumarin (AMC)-labeledpeptide substrates Acetyl-Tyr-Val-Ala-Asp-AMC (Ac-YVAD-AMC) andAcetyl-Asp-Glu-Val-Asp-pNA (Ac-DEVD-AMC). Ac-YVAD-pNA is describedfurther in Reiter, L. A. (1994) Int. J. Peptide Protein Res. 43:87-96.Ac-YVAD-pNA and Ac-YVAD-AMC are commercially available from BachemBioscience, Inc., King of Prussia, Pa. The rate constant of degradationof the modified ICE protein can be determined to quantitate thestability of the modified ICE protein.

[0022] Another aspect of the invention pertains to fusion proteins ofthe modified forms of ICE of the invention. The invention providesmodified ICE proteins that are fusion proteins. As used herein the term“fusion protein” refers to a modified form of ICE in which non-ICE aminoacid residues are fused at either the amino-terminus orcarboxy-terminus. A preferred modified ICE fusion protein is one thathas a polyhistidine tag (e.g., six histidine residues) at its aminoterminus. This polyhistidine fusion moiety allows for purification ofthe modified ICE protein on a nickel chelating column (Porath, J. (1992)Protein Expression and Purification 2:263-281). Other fusion moietiescan be used to facilitate protein purification, such asglutathione-S-transferase and maltose-binding protein. Preferably, amodified ICE fusion protein of the invention is prepared by recombinantDNA technology, as described further below.

[0023] II. Nucleic Acid Molecules Encoding Modified ICE Proteins

[0024] Another aspect of the invention pertains to nucleic acidmolecules encoding the modified ICE proteins of the invention. The aminoacid position that is modified in the ICE protein of the invention(i.e., position 381 of unmodified ICE), occurs within the p10 subunit ofmature ICE. Accordingly, the invention provides a nucleic acid moleculeencoding a modified p10 subunit of human ICE. This modified p10 subunitencoded by the nucleic acid comprises an amino acid sequence wherein anamino acid corresponding to aspartic acid at position 381 of unmodifiedICE (SEQ ID NO: 2) is replaced with a mutant amino acid, the mutantamino acid being capable of forming a salt bridge with an amino acidcorresponding to arginine at position 383 of unmodified ICE, such thatthe modified p10 subunit associates with a p20 subunit to form amodified ICE that retains proteolytic activity and exhibits increasedstability compared to unmodified ICE. Most preferably, the nucleic acidmolecule encodes a p10 subunit in which Asp-381 is replaced withglutamic acid. In other embodiments, the nucleic acid molecule encodes ap10 subunit in which Asp-381 is replaced with an amino acid selectedfrom the group consisting of serine, threonine, asparagine andglutamine.

[0025] As used herein, the term “nucleic acid molecule” is intended toinclude DNA molecules and RNA molecules. The nucleic acid molecule maybe single-stranded or double-stranded, but preferably is double-strandedDNA.

[0026] The modified ICE proteins and nucleic acid molecules of theinvention are preferably prepared using recombinant DNA technology. Forexample, to prepare a DNA fragment encoding a modified p10 subunit ofICE. first a DNA fragment encoding the region of unmodified ICEencompassing amino acid residues 317 to 404 (p10 ) is prepared, forexample, by PCR amplification using appropriate primers designed usingthe nucleotide sequence of ICE shown in SEQ ID NO: 1. Theoligonucleotide primers shown in SEQ ID NOs: 3 and 6 are suitable foramplifying a DNA fragment encoding an unmodified p10 subunit. The primerof SEQ ID NO: 3 corresponds to nucleotide sequences encoding the p10amino terminus (starting at amino acid 317) and contains an EcoRIrestriction site. The primer of SEQ ID NO: 6 is complementary tonucleotide sequences encoding the p10 carboxy terminus (amino acids400-404) followed by a stop codon and includes an SpeI restriction site.The DNA fragment encoding the unmodified ICE p10 subunit can then beused as a template for mutagenesis to create a DNA fragment encoding amodified ICE p10 subunit. Mutagenesis can be accomplished by PCRmutagenesis (as described further in Example 1), or other standardmethods known in the art such as site directed mutagenesis.

[0027] The nucleic acid molecules of the invention encoding modified ICEp10 subunits can be used to prepare modified ICE proteins of theinvention. The nucleic acid molecules can be incorporated intorecombinant expression vectors that allow for expression of the modifiedp10 subunit encoded therein. The modified p10 subunit can be expressedusing an in vitro transcription/translation system or, more preferably,is expressed by introducing the recombinant expression vector into asuitable host cell in which the p10 subunit is then expressed (e.g., E.coli). A recombinant expression vector of the invention can encode onlythe modified p10 ICE subunit (i.e., about amino acids 317-404 of ICE).Alternatively, the recombinant expression can encode both the modifiedp10 subunit and a p20 subunit (e.g., a wild-type p20 subunit, comprisingabout amino acids 120-297 of ICE)). For example, in one embodiment of arecombinant expression vector encoding both the p10 and p20 subunits,the vector carries a DNA fragment encoding amino acids 120 to 404 of ICE(referred to as p30), wherein the amino acid corresponding to Asp-381within the p10 subunit has been modified. The p30 fragment of ICE lacksthe “prodomain” from amino acids 1-119 but still contains the internalresidues 298-316, which are cleaved from the precursor form of ICE togenerate the p20 and p10 subunits. This p30 fragment is appropriatelyprocessed to p20 and p10 when expressed in a suitable host cell (e.g.,E. coli). In yet another embodiment of a recombinant expression vectorencoding both the p10 and p20 subunits, the vector encodes the entireprecurser form of ICE (i.e., amino acids 1-404), wherein the p10 subunithas been modified as described herein.

[0028] Accordingly, in one embodiment, a mature, active modified ICEprotein of the invention is prepared by coexpression of p20 and p10 in ahost cell (described further in Example 2, part B). The p20 and p10subunits can be coexpressed in a host cell using a p30-encodingexpression vector or, alternatively, by expression of separate p20 andp10 genes, either carried on the same vector or carried on two separateexpression vectors. In another embodiment, a mature, active modified ICEprotein of the invention is prepared by separately expressing the p20and p10 subunits in different host cell, recovering the two subunits,denaturing them and renaturing the two subunits together to form mature,active ICE comprised of associated p20 and p10 subunits (describedfurther in Example 2, part A).

[0029] III. Recombinant Expression Vectors

[0030] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a modified ICEp10 subunit of the invention, alone or together with a p20 subunit. Asused herein, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Onetype of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments may be ligated.Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are are referred to herein as “recombinant expressionvectors” or simply “expression vectors”. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids. In the present specification, “plasmid” and “vector” may beused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors, which serve equivalentfunctions.

[0031] In the recombinant expression vectors of the invention,ICE-encoding sequences are operatively linked to one or more regulatorysequences, selected on the basis of the host cells to be used forexpression. The term “operably linked” is intended to mean that theICE-encoding sequences are linked to the regulatory sequence(s) in amanner that allows for expression of ICE protein (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells thereby to produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

[0032] The recombinant expression vectors of the invention can bedesigned for expression of modified ICE proteins in prokaryotic oreukaryotic cells. For example, modified ICE proteins can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector may be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0033] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40 ), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. In apreferred embodiment, a DNA fragment encoding ICE p30 (amino acidresidues 120-404) is cloned into an expression vector (e.g., an E. coliexpression vector) that fuses a polyhistidine sequence (e g., sixhistidine residues) to the N-terminus of the p30-coding sequence. Thepolyhistidine fusion moiety allows for purification of the mature ICEprotein on a nickel chelating column (Porath, J. (1992) ProteinExpression and Purification 2:263-281). Polyhistidine fusion expressionvectors are commercially available (e.g., Novagen, Madison, Wis.).

[0034] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0035] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria that is impaired inits capacity to cleave proteolytically the recombinant protein(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 119-128). Another strategy isto alter the nucleic acid sequence of the nucleic acid to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in E. coli (Sharp and Li (1986)NucL. Acids Res., 14:7737-7749; Wada et al., (1992) NucL. Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

[0036] In another embodiment, the ICE expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

[0037] Alternatively, ICE can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al., (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

[0038] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

[0039] IV. Recombinant Host Cells

[0040] To prepare modified ICE proteins of the invention, typically oneor more recombinant expression vectors are introduced into a suitablehost cell in which the ICE protein is then expressed. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein. A host cell may be any prokaryotic or eukaryotic cell.For example, modified ICE proteins may be expressed in bacterial cellssuch as E. coli, insect cells, yeast or mammalian cells. Preferably,modified ICE proteins are expressed in E. coli as described in theExamples.

[0041] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

[0042] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker may be introduced into a host cell onthe same vector as that encoding ICE protein or may be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0043] V. Uses of Modified ICE Proteins

[0044] The modified ICE proteins of the invention retain the cysteineprotease activity of unmodified ICE. Accordingly, the modified ICEproteins are useful as cysteine proteases. Moreover, since a modifiedICE protein of the invention has increased stability compared tounmodified ICE, a particular amount of this modified ICE proteinexhibits greater proteolytic activity over time than an equal amount ofunmodified ICE. Another aspect of the invention, therefore, pertains tomethods for cleaving ICE substrates. The method involves contacting thesubstrate with a modified ICE protein of the invention such that the ICEsubstrate is cleaved. The term “ICE substrate” is intended to includeany peptide or protein which is cleavable by ICE. ICE substrates arecharacterized by an aspartic acid residue in the P1 position. Examplesof ICE substrates include prointerleukin-1β, the chromogenic pNA-labeledpeptide substrates Ac-YVAD-pNA and Ac-DEVD-pNA and the fluorogenicAMC-labeled peptide substrates Ac-YVAD-AMC and Ac-DEVD-AMC.

[0045] The modified ICE proteins of the invention can also be used inscreening assays to identify modulators of ICE activity. The inventionprovides a method for identifying a modulator of ICE protease activity,comprising:

[0046] preparing a modified ICE protein of the invention;

[0047] contacting the modified ICE with an ICE substrate in the presenceof a test compound under proteolytic conditions;

[0048] measuring proteolysis of the ICE substrate in the presence of thetest compound;

[0049] comparing proteolysis of the ICE substrate in the presence of thetest compound to proteolysis of the ICE substrate in the absence of thetest compound; and

[0050] identifying the test compound as a modulator of ICE proteaseactivity.

[0051] In one embodiment, the modified ICE protein comprises an aminoacid sequence wherein an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutantamino acid structure, the mutant amino acid structure being capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE, such that the modified ICE retainsenzymatic activity and exhibits increased stability compared tounmodified ICE. In the most preferred embodiment, the mutant amino acidstructure is glutamic acid. In other embodiments, the mutant amino acidstructure is selected from the group consisting of serine, threonine,asparagine and glutamine.

[0052] In one embodiment of the screening method, an inhibitor of ICEprotease activity is identified. In this case, the amount of proteolysisof the ICE substrate in the presence of the test compound is less thanthe amount of proteolysis of the ICE substrate in the absence of thetest compound. In another embodiment of the screening method, anactivator of ICE protease activity is identified. In this case, theamount of proteolysis of the ICE substrate in the presence of the testcompound is greater than the amount of proteolysis of the ICE substratein the absence of the test compound.

[0053] Suitable ICE substrates for use in the screening assays aredescribed above. Preferably, a chromogenic or fluorogenic ICE substrateis used whose cleavage can be detected spectrophotometrically.Alternatively, cleavage of other peptide substrate can be detectedchromatographically (e.g., by HPLC). Additionally, whole proteins can beused as substrates, such as prointerleukin-1β. Whole proteins can belabelled (e.g., with ³⁵S-methionine) and their cleavage products can bedirectly detected (e.g., by SDS-PAGE and autoradiography).Alternatively, cleavage of whole proteins can be detected indirectly(e.g., using an antibody that binds a specific cleavage product).

[0054] In addition to the foregoing uses, the modified ICE proteins ofthe invention, because of their enhanced stability, are particularlywell-suited for preparing crystalline ICE for X-ray crystallographicanalysis. For example, unmodified ICE labeled at its N-terminus with apolyhistidine tag and expressed in E. coli was found to be too unstable(i.e., underwent too much autodegradation) to prepare crystals thatcould be used for X-ray crystallography. In contrast, D381E-modified ICElabeled at its N-terminus with a polyhistidine tag and expressed in E.coli was sufficiently stable for the preparation of crystals for X-raycrystallography. Accordingly, modified ICE proteins of the invention canbe expressed recombinantly as described herein (e.g., in E. coli), therecombinant ICE protein can be purified and crystals can be preparedtherefrom for use in X-ray crystallography. General methods forpreparing crystalline ICE, and performing X-ray crystallographicanalysis thereon, are described in Walker, N. P. C. et al. (1994) Cell78:343-352 and Wilson, K. P. et al. (1994) Nature 370:270-275.Furthermore, a modified ICE protein of the invention can be used inX-ray crystallography in combination with a cysteine protease inhibitor,which serves to further stabilize the protein during crystallization, asdescribed in U.S. patent application Ser. No. ______, entitled “CysteineProtease Inhibitors and Uses Therefor”, filed on even date herewith andexpressly incorporated herein by reference.

[0055] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLE 1 Construction of a Modified ICE

[0056] A modified form of human ICE having an aspartic acid to glutamicacid substitution at amino acid position 381 (D381E) was constructed bypolymerase chain reaction (PCR) mutagenesis of a DNA fragment encodingamino acids 317-404 of ICE (i.e., the p10 subunit) followed by in vivorecombination in E. coli.

[0057] PCR Amplification

[0058] To create a DNA fragment encoding p10 (amino acids 317-404)having a D381E mutation, two different DNA fragments were amplified byPCR that together represent the ICE 317-404 gene with a 28 base pairoverlapping sequence (containing the mutation) where the two can belinked by homologous recombination in E. coli. Additionally, the DNAfragments were amplified such that they had restriction sites on eachend for cloning into an expression vector.

[0059] For the first fragment (referred to as Fragment A), a plasmidencoding wild-type ICE 317-404 having a methionine start codon insertedbefore the ICE alanine-317 was used as the template. This open readingframe had its codons modified to reflect the preferred codon usage ofhighly expressed genes in E. coli, according to the codon usageinformation disclosed in Sharp and Li (1986) Nucl. Acids Res.,14:7737-7749. The 5′ primer (#694) had the following sequence: 5′-GGGGAA TTC ATG GCT ATC AAA AAA GCT CAC ATC GAA AAA GAC TTC ATC GCT TTCTGC-3′ (SEQ ID NO: 3). This 5′ primer is complementary to the ICE317-404 amino terminus and contains an EcoRI restriction site. The 3′primer (#1537) had the following nucleotide sequence: 5′-TTC TGG CTG CTCAAA TGA AAA ACG AAC CTT GCG GAA AAT TTC-3′ (SEQ ID NO: 4). This 3′primer contains nucleotide sequences encoding the amino acid region ICE368-381 and contains 2 mutations. The first is an A to T point mutationat the 5′ end (position 1) of the oligonucleotide that changes asparticacid-381 to glutamic acid. The second is a silent point mutation (T to Aat position 22 of the oligo) that eliminates a TaqI site to be used forclone screening but does not further change the amino acid sequence ofthe encoded p10 subunit. PCR amplification using these two primers andthe wildtype ICE 317-404 containing plasmid as template resulted in afragment containing an open reading frame encoding ICE 317-381 havingthe D381E mutation, with an EcoRI site for cloning at the 5′ end.

[0060] For the second fragment (referred to as Fragment B), the templatewas the same as the first. The PCR reaction utilized a 5′ primer (#1533)having the nucleotide sequence: 5′-GGT TCG TTT TTC ATT TGA GCA GCC AGAAGG TAG AGC GCA GAT G-3′ (SEQ ID NO: 5). This 5′ primer containsnucleotide sequences encoding the amino acid region ICE 373-386 andcontains the complements to the 2 mutations described in the previousparagraph. The PCR reaction utilized a 3′ primer (#518) having thefollowing sequence: 5′-CCC CAC TAG TCC TCT ATT AAT GTC CTG GGA AGA GG-3′(SEQ ID NO: 6). The 3′ primer contains nucleotide sequences encoding theamino acid region ICE 400-404 followed by a stop codon and includes anSpel restriction site. PCR amplification using these primers and theplasmid encoding wild-type ICE 317-404 as template resulted in afragment containing an open reading frame encoding ICE 373-404,including the D381E mutation, followed at the 3′ end by the Spelrestriction site for cloning into the expression vector.

[0061] Standard PCR conditions were used to obtain the PCR fragmentsdescribed above using 30 cycles of 94° C. denaturing step for 30seconds, annealing for 30 seconds at 52° C. and 2 minutes elongation at72° C. These 30 cycles were followed by 5 minutes at 73° C. for finalextension. The reactions were then held at 4° C. until ready for furtheruse. Five microliters of each of the appropriate primers at 20 μMconcentrations were used in each reaction with the following additionalreaction components: 1 μl of template DNA at 1 μg/ml, 10 μl of standard10×PCR buffer containing MgCl₂ (PE Express, Norwalk, Conn.), 8 μl ofdNTP mix containing 2.5 mM each of deoxynucleotide triphosphate (PEExpress, Norwalk, Conn.), 0.5 μl Taq polymerase (PE Express, Norwalk,Conn.) and 70 μl of deionized dH₂O for a final volume of 100 μl.

[0062] 10 μl each of the two PCR fragments, Fragments A and B, from thetwo PCR reactions described above, were then separately run out on 1%agarose gels (FMC Bioproducts, Rockland, Me.) containing 0.5 μg/mlethidium bromide to visualize the resultant bands under UV Light. BothPCR products were observed, Fragment A at 206 base pairs and Fragment Bat 114 base pairs. The remaining PCR mix was extracted with an equalvolume of phenol:chloroform:isoamyl alcohol (25:24:1 v/v). The aqueousphase was brought to 1M LiCl₂ and the DNA was precipitated by theaddition of 3 volumes of 100% ethanol. The DNA was collected bycentrifugation. The pellet was washed with 70% ethanol, recentrifugedand dried under vacuum. The ends of the fragments were digested withEcoRI (fragment A) or SpeI (fragment B) in 50 μl reactions containingappropriate reaction buffer and 20 units of restriction enzyme. Thereactions were allowed to proceed at 37° C. overnight.

[0063] Following restriction enzyme digestion, Fragments A and B werethen separately electrophoresed on 4% Metaphor agarose gels (FMCBioproducts, Rocktand, Me.) containing 0.5 μg/ml ethidium bromide tovisualize the resultant bands under UV light. The expected size bandswere observed and purified by electrophoresis onto DEAE paper for 10minutes at 100 V and eluted with buffer containing 20% ethanol, 1 MLiCl₂, 10 mM tris pH 7.5, and 1 mM EDTA. The eluate was thenprecipitated with isopropanol, pelleted, washed with 70% ethanol, driedby speed vacuum and resuspended in 10 μl 10 mM tris, 1 mM EDTA pH 8.0.

[0064] Ligation of Fragments A and B to Expression Vector

[0065] The two PCR fragments (A and B) were ligated to the ends of thelinearized expression vector pJAM-4. pJAM-4 is a derivative ofpBluescript II KS(+) (Stratagene, La Jolla, Calif.) consisting of thebacteriophage Lambda left promoter (pL) followed by a ribosome bindingsite which is in turn followed by a polylinker sequence starting with anEcoRI site. The vector also carries an ampicillin resistance gene, theColE1 origin and the f1 (+) origin.

[0066] The expression vector pJAM-4 (10 μg) was digested with EcoRI andSpeI by standard methods. The linearized vector was purified from a 1%agarose gel containing 0.5 μg/ml ethidium bromide using DEAE paper, asdescribed above. The linearized vector was resuspended to an estimatedconcentration of 500 ng/μl in dH₂O.

[0067] A ligation reaction was prepared containing the followingreaction components: 5 μl each of fragments A and B, 1 μl of linearizedpJAM-4, 2 μl 10×Ligase buffer (New England BioLabs, Beverly, Mass.), 1μl T4 DNA Ligase (400 units) (New England BioLabs, Beverly, Mass.) and 6μl dH₂O. Ligation was carried out overnight at 16° C. Following theligation reaction, the ligation mix was extracted withphenol:chloroform:isoamyl alcohol (25:24:1 v/v) and precipitated asdescribed above. The resulting DNA pellet was dissolved in a finalvolume of 10 μl dH₂O.

[0068] Following the precipitation, the ligation products weretransformed into competent E coli strain MV1190 (Δ(lac-proAB), thi,supE, Δ(srl-recA) 306::Tn10(Tet^(r)) [F′:traD36, proAB, lacIq lacZΔM15])(Bio-Rad Laboratories, Hercules, Calif.) carrying plasmid pSE103. pSE103 is a kanamycin resistant pSC101 derivative carrying the temperaturesensitive bacteriophage Lambda repressor, cI⁸⁵⁷. Competent MV1190 pSE103cells were prepared as follows: A 3 ml seed culture in 2×YT media(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.:Cold Spring Harbor Press) was grown overnight at 30°C. from a frozen seed culture in the presence of 50 μug/ml kanamycin.The overnight culture was used to inoculate a larger culture (100-250 mlof 2×YT containing 50 μg/ml kanamycin) using a 1/50 dilution of the seedculture. The cells were grown with shaking at 30° C. until the OD₆₀₀value reached 0.5. Cells were harvested by low speed centrifugation, thecell pellet was resuspended in 1/2 original volume of ice cold 10 mMMgCl₂, 30 mM CaCl₂, 10% glycerol and the suspension allowed to sit onice for 30 minutes. The cells were recentrifuged at low speed and thecell pellet was resuspended in 1/10 the original culture volume of thesame solution above. The competent cells were dispensed into 100 μlaliquots and stored at −80° C. until use.

[0069] The cells were transformed using the linear DNA that resultedfrom the ligation of fragments A and B to the ends of the vector DNA, asfollows. In a pre-chilled tube, 5 μl of ligation mix was added to 100 μlof competent MV1190 pSE103 cells and incubated on ice for 30 minutes.The mixture was heat shocked at 37° C. for 2 minutes, then added to 1 ml2×YT medium and incubated for 1 hour at 30° C. 100 μl of the medium wasplated onto Luria-Bertani (LB) plates (ampicillin, 100 μg/ml; kanamycin,50 μg/ml) (Sambrook, et al., supra) and incubated at 30° C. overnight.The linear DNA, once inside the cell, undergoes a recA-independentrecombination event by virtue of the 28 base pair overlapping sequencesof Fragment A and Fragment B. This results in the recovery of an intactgene inserted into the expression vector which encodes p10 (amino acids317-404) having the D381E mutation.

[0070] Identification of Positive Clones

[0071] Transformants were screened by restriction enzyme digestion ofplasmid DNA minipreps. The enzymes used were EcoRI and HindIII.Transformants containing inserts of the correct size were used toprepare larger amounts of pure plasmid for further analysis. Thepresence of the D381E mutation was demonstrated by detecting the linkedmutation which resulted in the destruction of a TaqI site (by TaqIdigests) and by complete nucleotide sequence determination of theinsert.

[0072] Expression of ICE 317-404 D381E

[0073] Expression of the mutant p10 subunit (ICE 317-404 D381E) wascarried out in the protease-deficient E. coli strain CAG 597(F-;rpoH165(am) zhg::Tn10 lacZ(am) trp(am) pho(am) supCts mal(am) rpsL)(New England BioLabs, Beverly, Mass.) containing pACYC177cI⁸⁵⁷(Kanamycin^(R)). The p10 expression plasmid pJAM-4 ICE 317-404 D381E wastransformed into the bacterial cells as described above. Transformantswere selected on Luria-Bertani (LB) plates (ampicillin, 100 μg/ml;kanamycin, 50 μg/ml) (Sambrook, et al., supra) and incubated at 30° C.overnight. A single colony was used to inoculate a 3 ml 2×YT(ampicillin, 100 μug/ml; kanamycin, 50 μg/ml) culture and incubated at30° C. overnight. One liter of 2×YT (ampicillin, 100 μg/ml; kanamycin,50 μg/ml) was inoculated with 0.5 ml of overnight seed culture andincubated approximately 4 hours at 30° C. with shaking. Expression ofthe mutant p10 was induced by shifting the incubator temperature to 42°C. and the induced culture was incubated an additional 4 hours to allowfor protein production. The cells were harvested by centrifugation andthe pellets stored at −80° C. until ready for use.

EXAMPLE 2 Relative Activity and Stability of D381E-Modified ICE Comparedto Unmodified ICE

[0074] The D381E-modified form of ICE was prepared in two ways and itsactivity and/or stability was compared to wild-type (WT) ICE (i.e.,unmodified ICE). First, unmodified and D381 E-modified ICE were preparedby refolding separately-expressed p20 (WT) and p10 (WT or D381E mutant)subunits thereby to produce active, mature ICE (p20/p10). Second,unmodified and D381E-modified ICE were prepared by expression inbacteria of a single protein (p30) that encompasses both p20 and p10,and that autoproteolytically processes to the active, p20/p10 form(either fully WT or containing a p10 subunit carrying the D381Emutation). The p30-encoding constructs also contained an amino terminalpoly-Histidine tag to allow for rapid purification of the protein, andare referred to as WT N-His ICE (for wild-type) and D381E N-His ICE (forthe D381E mutant).

[0075] A. Comparative Activity of WT and D381E-Modified ICE Refoldedfrom p20 and p10 Subunits

[0076] ICE (WT or modified) was prepared by refolding p20 and p10, whichhad been separately expressed in E. coli, by a protocol described inU.S. patent application Ser. No. 08/242,663, entitled “ICE and ICE-LikeCompositions and Methods of Making Same”, which is expresslyincorporated herein by reference. The p20 subunit was prepared by HPLCpurification of material expressed as inclusion bodies in E. coli . Thep10 subunit (either wild-type, the D381A-modified form or theD381E-modified form) was used only as crude inclusion bodies. A controlof HPLC-purified wild-type p10 was also included to estimate the effecton the system of the crude inclusion bodies, but the conclusions of theexperiment were derived from the comparison of the three crudematerials.

[0077] During the final step of the refolding protocol, which consistsof dialysis from a tris buffer into a HEPES buffer, ICE proteolyticactivity against an ICE substrate develops gradually over a few hours.ICE activity was monitored as a function of time in the HEPES dialysis.Four samples, each differing in the p10 species used, were compared:HPLC-pure wild-type, crude wild-type, crude D381A, and crude D381E. Theresults of this experiment are shown below in Table I, wherein ICEactivity is expressed in arbitrary units. TABLE I Time into HEPESdialysis Pure WT Crude WT Crude D381A Crude D381E   0 hours  879  95  28 370   2 hours 2165 1200 237 3246 2.5 hours 4609 1332 220 3316 3.5 hours4789 1082 252 3976   4 hours 5228 1276 n.d. 3832 4.5 hours 4988 n.d.n.d. 3524   5 hours 5112 1356 584 4030

[0078] These results demonstrate that the D381E mutation produces amodified form of ICE with a final yield and/or specific activity that isequal to or greater than that of unmodified ICE. In contrast, the D381Amutation produces a modified form of ICE with substantially lowercatalytic activity than that of unmodified ICE.

[0079] B. Comparative Activity of Wild-type and D381E-Modified N-HisICE:

[0080] ICE (WT or modified) with an N-terminal polyhistidine tag wasexpressed in E. coli and purified using methodology described in Kamenset al. (1995) J. Biol. Chem., 270:15250-15256. Briefly, a DNA fragmentencoding amino acid residues 120 to 404 of wild-type or D381E-modifiedICE (p30) was cloned into an expression vector such that thep30-encoding fragment was fused in-frame at its N-terminus to DNAencoding six histidine residues. Following expression, the p30 fragmentis correctly processed to p20 and p10 subunits, which associate to formmature, active N-His-tagged ICE proteins. The N-His ICE fusion proteinsthen were purified on a nickel chelating column (Porath, J. (1992)Protein Expression and Purification 2:263-281).

[0081] The proteolytic activity of nickel-column-purified WT andD381E-modified ICE was examined using two pNA-labeled substrates,Ac-YVAD-pNA and Ac-DEVD-pNA. To perform the proteolysis assay, modifiedor unmodified N-His ICE was preincubated for 60 minutes at 30° C. in 80μl of a reaction buffer containing 100 mM HEPES, 20% (v/v) glycerol, 5mM DTT, 0.5 mM EDTA, at pH 7.5. The peptide substrate was added in 20 μlof reaction buffer containing 2.5 mM substrate and 5% DMSO solvent,giving final concentrations in the assay mixtures of 500 μM substrateand 1% DMSO. The incubation of N-His ICE with the peptide substrate at30° C. was continued, and the catalytic hydrolysis of peptide substratewas monitored by the change in absorbance of the samples at 405 nm dueto release of pNA as a function of time. Assays were performed induplicate. Samples were read using a microtiter plate reader (MolecularDevices, Sunnyvale, Calif.).

[0082] The Michaelis-Menten Km and Vmax values for the unmodified andmodified N-His ICE using the two peptide substrates were determined bystandard methods. The results are shown below: K_(m) values (μM) D381EWT Ac-YVAD-pNA 43 26 Ac-DEVD-pNA 44 25 V_(max) values (M ⁻¹ s⁻¹) D381EWT Ac-YVAD-pNA 0.19 0.04 Ac-DEVD-pNA 0.08 0.02

[0083] The similarities in the Km values for both substrates between thetwo enzymes demonstrate that WT and D381E-modified ICE are functionallysimilar. That the Vmax values are substantially greater with the D381Eform for both substrates, as compared to the WT form, most likelyreflects the fact that a large fraction of the WT enzyme is degraded andtherefore inactive.

[0084] C. Visualization/Quantitation of Increased Stability of D381EN-His ICE

[0085] To confirm that D381E-modified N-His ICE was more stable (i.e.,underwent less autocatalytic degradation) than WT N-His ICE, assuggested in part B above, nickel column purified material (WT or D381E)was analyzed by standard SDS-polyacrylamide electrophoresis.Representative results are shown in FIG. 1. The WT N-His ICE preparationconsisted of the expected p20 and p10 bands as well as smaller molecularweight breakdown products, the most visible being a p7 band in FIG. 1.By sequencing analysis, this p7 band has been identified as the largercleavage product that results from cleavage of p10 at Asp-381/Gly-382.Although not visible in FIG. 1, a p3 band, corresponding to the smallercleavage product that results from cleavage of p10 at Asp-281/Gly-382,has also been identified in the WT N-His ICE preparation. In contrast tothe WT preparation, these lower molecular weight breakdown products arenot detectable in the D381 E N-His ICE preparation shown in FIG. 1,whereas the p20 and p 10 subunits are readily detectable. The degree ofdegradation of p10 in the WT material is estimated to be at least 50 %compared to the modified material. This high degree of degradation ofthe WT sample rendered it unsuitable for use in crystallography. Incontrast, the D381E-modified material, which had no discernibledegradation of p10, was used successfully to form crystals that diffractto high resolution for X-ray crystallographic analysis of the ICEstructure.

[0086] Equivalents

[0087] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 6 1216 base pairs nucleic acid double linear cDNA CDS 1..1212 1 ATGGCC GAC AAG GTC CTG AAG GAG AAG AGA AAG CTG TTT ATC CGT TCC 48 Met AlaAsp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser 1 5 10 15 ATGGGC GAA GGT ACA ATA AAT GGC TTA CTG GAT GAA TTA TTA CAG ACA 96 Met GlyGlu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr 20 25 30 AGG GTGCTG AAC AAG GAA GAG ATG GAG AAA GTA AAA CGT GAA AAT GCT 144 Arg Val LeuAsn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala 35 40 45 ACA GTT ATGGAT AAG ACC CGA GCT TTG ATT GAC TCC GTT ATT CCG AAA 192 Thr Val Met AspLys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys 50 55 60 GGG GCA CAG GCATGC CAA ATT TGC ATC ACA TAC ATT TGT GAA GAA GAC 240 Gly Ala Gln Ala CysGln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp 65 70 75 80 AGT TAC CTG GCAGGG ACG CTG GGA CTC TCA GCA GAT CAA ACA TCT GGA 288 Ser Tyr Leu Ala GlyThr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95 AAT TAC CTT AAT ATGCAA GAC TCT CAA GGA GTA CTT TCT TCC TTT CCA 336 Asn Tyr Leu Asn Met GlnAsp Ser Gln Gly Val Leu Ser Ser Phe Pro 100 105 110 GCT CCT CAG GCA GTGCAG GAC AAC CCA GCT ATG CCC ACA TCC TCA GGC 384 Ala Pro Gln Ala Val GlnAsp Asn Pro Ala Met Pro Thr Ser Ser Gly 115 120 125 TCA GAA GGG AAT GTCAAG CTT TGC TCC CTA GAA GAA GCT CAA AGG ATA 432 Ser Glu Gly Asn Val LysLeu Cys Ser Leu Glu Glu Ala Gln Arg Ile 130 135 140 TGG AAA CAA AAG TCGGCA GAG ATT TAT CCA ATA ATG GAC AAG TCA AGC 480 Trp Lys Gln Lys Ser AlaGlu Ile Tyr Pro Ile Met Asp Lys Ser Ser 145 150 155 160 CGC ACA CGT CTTGCT CTC ATT ATC TGC AAT GAA GAA TTT GAC AGT ATT 528 Arg Thr Arg Leu AlaLeu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile 165 170 175 CCT AGA AGA ACTGGA GCT GAG GTT GAC ATC ACA GGC ATG ACA ATG CTG 576 Pro Arg Arg Thr GlyAla Glu Val Asp Ile Thr Gly Met Thr Met Leu 180 185 190 CTA CAA AAT CTGGGG TAC AGC GTA GAT GTG AAA AAA AAT CTC ACT GCT 624 Leu Gln Asn Leu GlyTyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 TCG GAC ATG ACTACA GAG CTG GAG GCA TTT GCA CAC CGC CCA GAG CAC 672 Ser Asp Met Thr ThrGlu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 AAG ACC TCT GACAGC ACG TTC CTG GTG TTC ATG TCT CAT GGT ATT CGG 720 Lys Thr Ser Asp SerThr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240 GAA GGC ATTTGT GGG AAG AAA CAC TCT GAG CAA GTC CCA GAT ATA CTA 768 Glu Gly Ile CysGly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255 CAA CTC AATGCA ATC TTT AAC ATG TTG AAT ACC AAG AAC TGC CCA AGT 816 Gln Leu Asn AlaIle Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270 TTG AAG GACAAA CCG AAG GTG ATC ATC ATC CAG GCC TGC CGT GGT GAC 864 Leu Lys Asp LysPro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280 285 AGC CCT GGTGTG GTG TGG TTT AAA GAT TCA GTA GGA GTT TCT GGA AAC 912 Ser Pro Gly ValVal Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290 295 300 CTA TCT TTACCA ACT ACA GAA GAG TTT GAG GAT GAT GCT ATT AAG AAA 960 Leu Ser Leu ProThr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys 305 310 315 320 GCC CACATA GAG AAG GAT TTT ATC GCT TTC TGC TCT TCC ACA CCA GAT 1008 Ala His IleGlu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp 325 330 335 AAT GTTTCT TGG AGA CAT CCC ACA ATG GGC TCT GTT TTT ATT GGA AGA 1056 Asn Val SerTrp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg 340 345 350 CTC ATTGAA CAT ATG CAA GAA TAT GCC TGT TCC TGT GAT GTG GAG GAA 1104 Leu Ile GluHis Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 355 360 365 ATT TTCCGC AAG GTT CGA TTT TCA TTT GAG CAG CCA GAT GGT AGA GCG 1152 Ile Phe ArgLys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala 370 375 380 CAG ATGCCC ACC ACT GAA AGA GTG ACT TTG ACA AGA TGT TTC TAC CTC 1200 Gln Met ProThr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu 385 390 395 400 TTCCCA GGA CAT TAAA 1216 Phe Pro Gly His 404 amino acids amino acid linearprotein 2 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile ArgSer 1 5 10 15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu LeuGln Thr 20 25 30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg GluAsn Ala 35 40 45 Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val IlePro Lys 50 55 60 Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys GluGlu Asp 65 70 75 80 Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp GlnThr Ser Gly 85 90 95 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu SerSer Phe Pro 100 105 110 Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met ProThr Ser Ser Gly 115 120 125 Ser Glu Gly Asn Val Lys Leu Cys Ser Leu GluGlu Ala Gln Arg Ile 130 135 140 Trp Lys Gln Lys Ser Ala Glu Ile Tyr ProIle Met Asp Lys Ser Ser 145 150 155 160 Arg Thr Arg Leu Ala Leu Ile IleCys Asn Glu Glu Phe Asp Ser Ile 165 170 175 Pro Arg Arg Thr Gly Ala GluVal Asp Ile Thr Gly Met Thr Met Leu 180 185 190 Leu Gln Asn Leu Gly TyrSer Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 Ser Asp Met Thr ThrGlu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 Lys Thr Ser AspSer Thr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240 Glu GlyIle Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255 GlnLeu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290295 300 Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys305 310 315 320 Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser ThrPro Asp 325 330 335 Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val PheIle Gly Arg 340 345 350 Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser CysAsp Val Glu Glu 355 360 365 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu GlnPro Asp Gly Arg Ala 370 375 380 Gln Met Pro Thr Thr Glu Arg Val Thr LeuThr Arg Cys Phe Tyr Leu 385 390 395 400 Phe Pro Gly His 57 bases nucleicacid single linear oligonucleotide primer 3 GGGGAATTCA TGGCTATCAAAAAAGCTCAC ATCGAAAAAG ACTTCATCGC TTTCTGC 57 42 bases nucleic acid singlelinear oligonucleotide primer 4 TTCTGGCTGC TCAAATGAAA AACGAACCTTGCGGAAAATT TC 42 43 bases nucleic acid single linear oligonucleotideprimer 5 GGTTCGTTTT TCATTTGAGC AGCCAGAAGG TAGAGCGCAG ATG 43 35 basesnucleic acid single linear oligonucleotide primer 6 CCCCACTAGTCCTCTATTAA TGTCCTGGGA AGAGG 35

1. A modified interleukin-1β converting enzyme (ICE) comprising an aminoacid sequence wherein an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutantamino acid structure, the mutant amino acid structure being capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE, such that the modified ICE retainsproteolytic activity and exhibits increased stability compared tounmodified ICE.
 2. The modified ICE of claim 1, wherein the mutant aminoacid structure is a natural amino acid.
 3. The modified ICE of claim 1,wherein the mutant amino acid structure is a non-natural amino acid. 4.The modified ICE of claim 1, which is at least 25% more stable thanunmodified ICE.
 5. The modified ICE of claim 1, which is at least 50%more stable than unmodified ICE.
 6. The modified ICE of claim 1, whichis a fusion protein.
 7. A modified interleukin-1β converting enzyme(ICE) comprising an amino acid sequence wherein an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE (SEQ IDNO: 2) is replaced with glutamic acid.
 8. The modified ICE of claim 7,which is a fusion protein.
 9. A modified interleukin-1β convertingenzyme (ICE) comprising an amino acid sequence wherein an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE (SEQ IDNO: 2) is replaced with an amino acid selected from the group consistingof serine, threonine, asparagine and glutamine.
 10. The modified ICE ofclaim 9, which is a fusion protein.
 11. A nucleic acid molecule encodinga modified p10 subunit of interleukin-1β converting enzyme (ICE), themodified p10 subunit comprising an amino acid sequence wherein an aminoacid corresponding to aspartic acid at position 381 of unmodified ICE(SEQ ID NO: 2) is replaced with a mutant amino acid structure, themutant amino acid structure being capable of forming a salt bridge withan amino acid corresponding to arginine at position 383 of unmodifiedICE, such that the modified p10 subunit associates with a p20 subunit toform a modified ICE which retains proteolytic activity and exhibitsincreased stability compared to unmodified ICE.
 12. A recombinantexpression vector comprising the nucleic acid molecule of claim
 11. 13.The recombinant expression vector of claim 12, which further encodes ap20 subunit of ICE.
 14. A host cell containing the recombinantexpression vector of claim
 12. 15. A host cell containing therecombinant expression vector of claim
 13. 16. A nucleic acid moleculeencoding a modified p10 subunit of interleukin-1β converting enzyme(ICE), the modified p10 subunit comprising an amino acid sequencewherein an amino acid corresponding to aspartic acid at position 381 ofunmodified ICE (SEQ ID NO: 2) is replaced with glutamic acid.
 17. Arecombinant expression vector comprising the nucleic acid molecule ofclaim
 16. 18. The recombinant expression vector of claim 17, whichfurther encodes a p20 subunit of ICE.
 19. A host cell containing therecombinant expression vector of claim
 17. 20. A host cell containingthe recombinant expression vector of claim
 18. 21. A nucleic acidmolecule encoding a modified p10 subunit of interleukin-1β convertingenzyme (ICE), the modified p10 subunit comprising an amino acid sequencewherein an amino acid corresponding to aspartic acid at position 381 ofunmodified ICE (SEQ ID NO: 2) is replaced with an amino acid selectedfrom the group consisting of serine, threonine, asparagine andglutamine.
 22. A recombinant expression vector comprising the nucleicacid molecule of claim
 21. 23. The recombinant expression vector ofclaim 22, which further encodes a p20 subunit of ICE.
 24. A host cellcontaining the recombinant expression vector of claim
 22. 25. A hostcell containing the recombinant expression vector of claim
 23. 26. Amethod for cleaving an interleukin-1β converting enzyme (ICE) substrate,comprising contacting the substrate with a modified ICE such that theICE substrate is cleaved, wherein the modified ICE comprises an aminoacid sequence having an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) replaced with a mutantamino acid structure, the mutant amino acid structure being capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE.
 27. The method of claim 26, wherein themutant amino acid structure is glutamic acid.
 28. The method of claim26, wherein the mutant amino acid structure is selected from the groupconsisting of serine, threonine, asparagine and glutamine.
 29. A methodfor identifying a modulator of interleukin-1β converting enzyme (ICE)protease activity, comprising: preparing a modified ICE comprising anamino acid sequence wherein an amino acid corresponding to aspartic acidat position 381 of unmodified ICE (SEQ ID NO: 2) is replaced with amutant amino acid structure, the mutant amino acid structure beingcapable of forming a salt bridge with an amino acid corresponding toarginine at position 383 of unmodified ICE, such that the modified ICEretains proteolytic activity and exhibits increased stability comparedto unmodified ICE; contacting the modified ICE with an ICE substrate inthe presence of a test compound under proteolytic conditions; measuringproteolysis of the ICE substrate in the presence of the test compound;comparing proteolysis of the ICE substrate in the presence of the testcompound to proteolysis of the ICE substrate in the absence of the testcompound; and identifying the test compound as a modulator of ICEprotease activity.
 30. The method of claim 29, wherein the mutant aminoacid structure is glutamic acid.
 31. The method of claim 29, wherein themutant amino acid structure is selected from the group consisting ofserine, threonine, asparagine and glutamine.
 32. The method of claim 29,wherein the test compound inhibits ICE protease activity.
 33. The methodof claim 29, wherein the test compound stimulates ICE protease activity.